Enhanced vaccines

ABSTRACT

The invention relates to methods and materials involved in the treatment and prevention of various diseases such as infections and IgE-related diseases. Specifically, the invention relates to methods and materials that can be used to vaccinate a mammal against specific self or non-self antigens. For example, the methods and materials described herein can be used to reduce the effects of IgE antibodies within a mammal by reducing the amount of total and receptor bound IgE antibodies in the mammal. In addition, the invention provides vaccine conjugates, immunogenic polypeptides, nucleic acid molecules that encode immunogenic polypeptides, host cells containing the nucleic acid molecules that encode immunogenic polypeptides, and methods for making vaccine conjugates and immunogenic polypeptides as well as nucleic acid molecules that encode immunogenic polypeptides. Further, the invention provides an IgE vaccine that induces an anti-self IgE response in a mammal.

RELATED APPLICATIONS

[0001] This application claims priority from U.S. ProvisionalApplication Serial No. 60/106,652, filed Nov. 2, 1998.

BACKGROUND

[0002] 1. Technical Field

[0003] The invention relates to methods and materials involved in thetreatment of various diseases such as infections and IgE-relateddiseases. Specifically, the invention relates to methods and materialsthat can be used to vaccinate a mammal against specific self or non-selfantigens. For example, the methods and materials described herein can beused to reduce the effects of IgE antibodies within a mammal.

[0004] 2. Background Information

[0005] Mammals are susceptible to many diseases and illnesses includingbacterial infections, viral infections, and IgE-related diseases such asallergies. In general, infections are characterized by the invasion andmultiplication of microorganisms (e.g., bacteria, fungi, and viruses)within body tissues. Many types of infections can be treated orprevented by the use of vaccines. For example, the polio vaccine canprevent poliovirus infections. Typically, a vaccine is a suspension ofattenuated or killed microorganisms.

[0006] IgE-related diseases are mediated by a class of immunoglobulindesignated as immunoglobulin E (IgE). In fact, IgE antibodies are amajor cause of hypersensitivity reactions found within the humanpopulation despite their normally very low concentration in human plasma(10-400 ng/mL). The effects are due to the interaction of IgE antibodieswith the high-affinity receptor for IgE on mast cells and basophilicleukocytes. Cross-linking of two IgE receptors on the surface of thesecell types, for example by allergen binding, initiates the release of anumber of physiologically active substances such as histamine, PAF(platelet activating factor), heparin, leukotrienes, prostaglandins,thromboxanes, and chemotactic factors for eosinophilic and neutrophilicgranulocytes. Presumably, these mediators cause the direct symptoms ofIgE-mediated allergic reactions (type I hypersensitivity). Diseaseconditions belonging to this-group can include asthma, fur allergies,pollen allergies, food allergies, and eczema.

[0007] The high-affinity receptor for IgE has been characterized. Thisreceptor appears to be present on mast cells, basophilic leucocytes,eosinophils, monocytes, and Langerhan cells. In addition, the receptoris a complex of three different subunits (α, β, and γ chains). The αchain is localized mainly extra-cellularly and appears to interact withthe IgE molecule. Previous studies of the epsilon chain of the IgEmolecule have suggested that a region of 76 amino acids at the borderbetween the CH2 and CH3 domains (CH refers to the constant domains inthe heavy chain) is important for the interaction between the IgEmolecule and its high-affinity receptor. In addition, a peptidecorresponding to this region was shown to inhibit the interactionbetween native IgE and its high-affinity receptor in vitro at a molarratio of nearly 1:1 compared to the whole CH2-CH3-CH4 region (Helm etal., Nature 331, 180-183 (1988)). This peptide was also shown to inhibitan IgE-mediated flare reaction in allergen stimulation. In this case,however, the concentration was about 10 times the concentration neededto exhibit the same inhibitory effect with native IgE (Helm et al.,Proc. Natl. Acad Sci. USA 86, 9465-9469 (1989)).

SUMMARY

[0008] The invention relates to methods and materials involved in thetreatment and prevention of various diseases such as infections andIgE-related diseases. Specifically, the invention relates to methods andmaterials that can be used to vaccinate a mammal against specific selfor non-self antigens. For example, the methods and materials describedherein can be used to reduce the effects of IgE antibodies within amammal by reducing the amount of total and receptor bound IgE antibodiesin the mammal. Such methods and materials can be used to treat atopicallergies in mammals such as humans, dogs, and pigs.

[0009] The invention is based on the discovery that a vaccine conjugatecan be designed to contain at least two polypeptides with eachpolypeptide having at least two similar amino acid segments such thatthe administration of the conjugate to a mammal can induce an immuneresponse against at least a portion of one of the polypeptides. Suchimmune responses can be more potent than the responses induced by any ofthe polypeptides in an unconjugated form or any conjugate ofpolypeptides lacking at least two similar amino acid segments. Thus, thevaccine conjugates described herein can be used to provide mammals withsubstantial protection against a wide range of either self (e.g., IgEmolecules) or non-self (e.g., viral polypeptides) antigens.

[0010] The invention also is based on the discovery that a vaccineconjugate can be designed to contain a polypeptide having a cytokineactivity such that a potent immune response is induced against anotherpolypeptide within the conjugate. Such immune responses can be morepotent than the responses induced by a conjugate lacking a polypeptidehaving a cytokine activity. Although not limited to any particular modeof action, a conjugate containing a polypeptide having a cytokineactivity as well as an immunogenic polypeptide presumably concentratescytokine activity to the localized area containing the immunogenicpolypeptide. Thus, the polypeptide having cytokine activity canstimulate cells that participate in generating a specific immuneresponse against the immunogenic polypeptide.

[0011] In addition, the invention is based on the discovery thatpolypeptides containing a self IgE portion and a non-self IgE portionare immunogenic and induce an effective anti-self IgE response inmammals. Such immunogenic polypeptides can be used as a vaccine toinduce an anti-self IgE response that counteracts the hypersensitivityinduced by self IgE antibodies. Although not limited to any particularmode of action, the immunogenic polypeptides described herein induce theproduction of anti-self IgE antibodies that presumably have specificityfor the portion of the IgE molecule that interacts with thehigh-affinity IgE receptor. After production, the anti-self IgEantibodies can interact with the self-IgE antibodies such that the selfIgE antibodies are unable to bind to the high-affinity IgE receptor.This inhibition of receptor binding presumably reduces thehypersensitivity induced by self IgE antibodies. Thus, the degree ofIgE-induced effects can be reduced as more anti-self IgE antibodies areproduced.

[0012] In general, the invention features an immunogenic polypeptidehaving a self IgE portion and a non-self IgE portion. The immunogenicpolypeptide is effective to induce an anti-self IgE response in a mammal(e.g., human). The self portion can contain at least a portion of a CH3domain of IgE. The polypeptide can be capable of dimerizing to form asoluble immunogenic dimer effective to induce the anti-self IgE responsein the mammal. The non-self IgE portion can contain a first region and asecond region with the self IgE portion being located between the firstand second regions of the non-self IgE portion. The first region cancontain at least a portion of an IgE CH2 domain, and the second regioncan contain at least a portion of an IgE CH4 domain. The non-self IgEportion can contain an IgE sequence present in a non-placental mammal(e.g., opossum, platypus, koala, kangaroo, wallaby, and wombat). Theself IgE portion can lack the CH2 domain of an IgE antibody. Theimmunogenic polypeptide can contain a eukaryotic post-translationalmodification. In addition, the immunogenic polypeptide can contain apolyhistidine sequence. The anti-self IgE response can be a polyclonalresponse.

[0013] In another embodiment, the invention features a nucleic acidmolecule containing a nucleic acid sequence that encodes an immunogenicpolypeptide. The immunogenic polypeptide contains a self IgE portion aswell as a non-self IgE portion, and is effective to induce an anti-selfIgE response in a mammal. The nucleic acid molecule can contain anadditional nucleic acid sequence that encodes an amino. acid sequencethat promotes the secretion of the immunogenic polypeptide from aeukaryotic cell.

[0014] Another embodiment of the invention features a host cell (e.g.,eukaryotic cell) containing a nucleic acid molecule that has a nucleicacid sequence that encodes an immunogenic polypeptide. The immunogenicpolypeptide contains a self IgE portion as well as a non-self IgEportion, and is effective to induce an anti-self IgE response in amammal.

[0015] Another embodiment of the invention features a solubleimmunogenic dimer containing two immunogenic polypeptides that arecapable of dimerizing to form the soluble immunogenic dimer. Each of thetwo immunogenic polypeptides contains a self IgE portion and a non-selfIgE portion, and the soluble immunogenic dimer is effective to induce ananti-self IgE response in a mammal.

[0016] Another embodiment of the invention features a vaccine containingan immunogenic polypeptide having a self IgE portion and a non-self IgEportion. The immunogenic polypeptide is effective to induce an anti-selfIgE response in a mammal. The vaccine can contain a pharmaceuticallyacceptable carrier.

[0017] Another embodiment of the invention features a method for makinga nucleic acid molecule that encodes an immunogenic polypeptideeffective to induce an anti-self IgE response in a mammal. The methodincludes combining first and second nucleic acid sequences to form thenucleic acid molecule, where the first nucleic acid sequence encodes atleast a portion of an IgE molecule present within the mammal, and wherethe second nucleic acid sequence encodes at least a portion of an IgEmolecule not present in the mammal.

[0018] Another embodiment of the invention features a method for makinga nucleic acid molecule that encodes an immunogenic polypeptideeffective to induce an anti-self IgE response in a mammal. The methodincludes (a) selecting a first nucleic acid sequence, where the firstnucleic acid sequence encodes at least a portion of an IgE moleculepresent within the mammal, (b) selecting a second nucleic acid sequence,where the second nucleic acid sequence encodes at least a portion of anIgE molecule not present in the mammal, and (c) combining the first andsecond nucleic acid sequences to form the nucleic acid molecule.

[0019] In another aspect, the invention features a vaccine complex forvaccinating a mammal (e.g., human). The complex contains a first andsecond polypeptide. Each of the first and second polypeptides containsat least two similar amino acid sequences at least five amino acidresidues in length. In addition, the first and second polypeptides areconnected to form the complex, and administration of the complex to themammal induces an immune response against at least a portion of thefirst or second polypeptide. The first and/or second polypeptide cancontains an amino acid sequence expressed by the mammal. The first andsecond polypeptides can be identical, and can form a dimer. Theconnection of the first and second polypeptides can include a disulfidebond. The connection of the first and second polypeptides can include anon-covalent interaction. The first and/or second polypeptide cancontain a linker site (e.g., a polyhistidine sequence). The amino andcarboxyl termini of the first and/or second polypeptide can contain thelinker site. The complex can include a linking molecule (e.g., anantibody such as an anti-polyhistidine antibody). A linking molecule canconnects the first and second polypeptide. The complex can contain athird polypeptide, where the third polypeptide has a cytokine activity.The cytokine activity can be an activity of a cytokine such asinterferon-α, interferon-β, interferon-γ, TNF-α, IL-1, IL-2, IL-4, IL-6,IL-12, IL-15, IL-18, and granulocyte-macrophage colony stimulatingfactor. A linking molecule can connect the third polypeptide to thefirst or second polypeptide. The similar amino acid sequences can begreater than about twenty amino acid residues in length. The complex cancontain an Fc-gamma receptor II blocking molecule (e.g., an anti-CD32antibody).

[0020] In another embodiment, the invention features a vaccine complexfor vaccinating a mammal (e.g., human). The complex contains a firstpolypeptide connected to a second polypeptide, where the firstpolypeptide contains at least two similar amino acid sequences at leastfive amino acids in length. In addition, the second polypeptide has acytokine activity, and administration of the complex to the mammalinduces an immune response against at least a portion of the firstpolypeptide. The first polypeptide can contain an amino acid sequenceexpressed by the mammal. The connection of the first and secondpolypeptides can include a non-covalent interaction. The first and/orsecond polypeptide can contain a linker site (e.g., a polyhistidinesequence). For example, the amino and carboxyl termini of the firstpolypeptide can contain a linker site. The complex can contain a linkingmolecule (e.g., an antibody such as an anti-polyhistidine antibody). Thecytokine activity can be an activity of a cytokine such as interferon-α,interferon-β, interferon-γ, TNF-α, IL-1, IL-2, IL-4, IL-6, IL-12, IL-15,IL-18, and granulocyte-macrophage colony stimulating factor. The complexcan contain a third polypeptide. The first and third polypeptides can beidentical and can form a dimer. The connection of the first and thirdpolypeptides can include a disulfide bond. The similar amino acidsequences can be greater than about twenty amino acid residues inlength. The complex can contain an Fc-gamma receptor II blockingmolecule (e.g., an anti-CD32 antibody).

[0021] Another embodiment of the invention features a vaccine complexfor vaccinating a mammal (e.g., human). The complex contains a first,second, and third polypeptide, where the first, second, and thirdpolypeptides are connected to form the complex. The first polypeptidehas a first cytokine activity. The second polypeptide has a secondcytokine activity. The administration of the complex to the mammalinduces an immune response against at least a portion of the thirdpolypeptide. The third polypeptide can contain an amino acid sequenceexpressed by the mammal. The connections of the first, second, and thirdpolypeptides can include non-covalent interactions. The first, second,and/or third polypeptide can contain a linker site. The complex cancontain a linking molecule. The third polypeptide can contain at leasttwo similar amino acid sequences at least five amino acids in length.The complex can contain an Fc-gamma receptor II blocking molecule (e.g.,an anti-CD32 antibody).

[0022] Another embodiment of the invention features a vaccine complexfor vaccinating a mammal (e.g., human). The complex contains a firstpolypeptide connected to a second polypeptide, where the firstpolypeptide is a polypeptide having interferon-α or interferon-βactivity, and administration of the complex to the mammal induces animmune response against at least a portion of the second polypeptide.The second polypeptide can contain an amino acid sequence expressed bythe mammal. The connection of the first and second polypeptides caninclude a non-covalent interaction. The first and/or second polypeptidecan contain a linker site. The complex can contain a linking molecule.The second polypeptide can contain at least two similar amino acidsequences at least five amino acids in length. The complex can containan Fc-gamma receptor II blocking molecule (e.g., an anti-CD32 antibody).

[0023] Another aspect of the invention features a vaccine forvaccinating a mammal (e.g., human). The vaccine contains an Fc-gammareceptor II blocking molecule (e.g., an anti-CD32 antibody) and apolypeptide, where administration of the vaccine to the mammal inducesan immune response against at least a portion of the polypeptide. Thepolypeptide can contain an amino acid sequence expressed by the mammal.The Fc-gamma receptor II blocking molecule and polypeptide can beconnected, and the connection can include a non-covalent interaction.

[0024] Unless otherwise defined, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

[0025] Other features and advantages of the invention will be apparentfrom the following detailed description, and from the claims.

DESCRIPTION OF DRAWINGS

[0026]FIG. 1 is a diagram comparing the amino acid sequences of theCH2-CH3-CH4 domains of human, rat, and opossum IgE, in the upper,middle, and lower rows, respectively. The opossum sequence also containsan N-terminal signal sequence followed by six histidine residues.

[0027] FIGS. 2A-B contain diagrams comparing the amino acid sequences ofvarious polypeptides containing the following components: opossumCH2—rat CH3—opossum CH4 (ORO); opossum-CH2—rat N-term CH3—opossum C-termCH3—opossum CH4 (ORO-trunc); opossum CH2—mouse CH3—opossum CH4 (OMO);opossum CH2—CH3—CH4 (OOO); platypus CH2—CH3—CH4 (PPP); opossum CH2—humanCH3—opossum CH4 (OHO); opossum CH2—pig CH3—opossum CH4 (OPO); andopossum CH2—dog CH3—opossum CH4 (ODO). The arrows indicate domainborders.

[0028] FIGS. 3A-C contain diagrams depicting the analysis of immuneresponses against an ORO immunogenic polypeptide in a panel of threedifferent strains of rats. The level of rat IgG anti-IgE antibodiesdirected against native rat IgE was measured by an ELISA. Native rat IgEwas used at a concentration of 5 μg/mL for coating of ELISA plates.Successive 1/5 dilutions of serum from each of the individual rats weretested by color reaction in the ELISA. Six vaccinated rats were analyzedtogether with four control rats from each strain.

[0029]FIG. 4 is a diagram depicting an analysis of the immune responsesagainst an ORO immunogenic polypeptide as well as OOO and PPP controlpolypeptides.

DETAILED DESCRIPTION

[0030] The invention provides methods and materials for the treatment ofvarious diseases such as infections and IgE-related diseases.Specifically, the invention provides methods and materials that can beused to vaccinate a mammal against specific self or non-self antigens.For example, the methods and materials described herein can be used toreduce the effects of IgE antibodies within a mammal by reducing theamount of total and receptor bound IgE antibodies in the mammal.

[0031] 1. Vaccine Conjugates

[0032] The invention provides vaccine conjugates that contain at leasttwo polypeptides with each of those polypeptides having at least twosimilar amino acid segments. The term “conjugate” as used herein refersto any composition containing at least two polypeptides that aredirectly or indirectly connected via one or more covalent ornon-covalent bonds. For example, a conjugate can contain tensequentially connected polypeptides (e.g., number one is connected tonumber two, number two is connected to number three, number three isconnected to number four, etc.). The term “connected” as used hereinwith respect to polypeptides refers to any type of covalent ornon-covalent bond including, without limitation, single bonds, doublebonds, triple bonds, disulfide bonds, hydrogen bonds, hydrophobicinteractions, van der Waals interactions, and any combination thereof.For example, a disulfide bond can connect polypeptide number one topolypeptide number two. Alternatively, an antibody can connectpolypeptide numbers one and two. In this case, polypeptides one and twoeach would contain an epitope recognized by the antibody such that theresulting conjugate contains polypeptide number one non-covalentlyconnected to the antibody which is non-covalently connected topolypeptide number two. It is noted that polypeptide numbers one and twoin this example can have an identical amino acid sequence.

[0033] The term “amino acid segment” as used herein refers to acontiguous stretch of amino acid sequence within a polypeptide. Forexample, the amino acid sequence from residues 30 to 40 within a 100amino acid polypeptide would be considered an amino acid segment. Forthe purpose of this invention, an amino acid segment can be any lengthgreater than about five amino acid residues (e.g., greater than aboutsix, seven, eight, nine, ten, 15, 20, 25, 30, 40, 50, 75, 100, 150, or200 amino acid residues). Thus, an amino acid segment can be the entireCH3 domain of an IgE antibody.

[0034] The term “similar” as used herein with respect to at least twoamino acid segments means the segments are at least about 50 percentidentical in amino acid sequence. For example, similar amino acidsegments can be about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or 100percent identical. For the purpose of this invention, the percent aminoacid sequence identity between one amino acid segment and another iscalculated as follows. First, the amino acid sequences of the two aminoacid segments are aligned using the MEGALIGN® (DNASTAR, Madison, Wis.,1997) sequence alignment software following the Jotun Heim algorithmwith the default settings. Second, the number of matched positionsbetween the two aligned amino acid sequences is determined. A matchedposition refers to a position in which identical residues occur at thesame position as aligned by the MEGALIGN® sequence alignment software.Third, the number of matched positions is divided by the total number ofpositions, and the resulting value multiplied by 100 to obtain thepercent identity.

[0035] Again, a vaccine conjugate of the invention contains at least twopolypeptides with each of those polypeptides having at least two similaramino acid segments. Thus, a vaccine conjugate can contain two, three,four, five, six, seven, eight, nine, ten, 15, 20, 25, or 30 polypeptideswith each having at least two similar amino acid segments. It is notedthat a polypeptide containing at least two similar amino acid segmentscan contain two, three, four, five, six, seven, eight, nine, ten, ormore similar amino acid segments. In addition to the polypeptidescontaining at least two similar amino acid segments, a vaccine conjugateof the invention can contain any number of polypeptides not having atleast two similar amino acid segments. For example, a vaccine conjugatecan contain four polypeptides each having a 30 amino acid residuesegment repeated three times as well as two polypeptides each lackingsimilar amino acid segments.

[0036] Typically, a vaccine conjugate contains a polypeptide that willact as an antigen against which an immune response is desired. Thus, avaccine conjugate within the scope of the invention can contain any typeof polypeptide including, without limitation, bacterial polypeptides,fungal polypeptides, viral polypeptides, and mammalian polypeptides. Forexample, a vaccine conjugate can contain five hepatitis C viruspolypeptides. It is noted that each polypeptide of a conjugate can havean identical amino acid sequence. In addition, a polypeptide of avaccine conjugate typically contains similar amino acid segments each ofwhich can act as a defined antigenic unit against which an immuneresponse is desired. Thus, a polypeptide of a vaccine conjugate cancontain similar amino acid segments that correspond to any region from apolypeptide including, without limitation, receptor binding regions,ligand binding regions, enzyme active sites, enzyme cleavage sites ofpolypeptide substrates, antigen-binding regions of antibodies, andepitopes recognized by antibodies. For example, a polypeptide of avaccine conjugate can contain three similar amino acid segments thateach correspond to the enzyme active site of enzyme X. It is noted thatsimilar amino acid segments can be in tandem or dispersed throughout apolypeptide. Typically, the administration of a vaccine conjugateresults in the formation of antibodies having specificity for an epitopeformed by at least a portion of the similar amino acid segments withinone of the polypeptides of the vaccine conjugate.

[0037] Any method can be used to make the polypeptides of a vaccineconjugate including, without limitation, prokaryotic expression systems,eukaryotic expression systems, and chemical synthesis techniques. Inaddition, a polypeptide of a vaccine conjugate can be obtained fromnatural tissue sources. For example, a brain glycopolypeptide can beobtained from brain tissue. Typically, each different polypeptide of aconjugate is made independently, or isolated independently, and thenused to form a conjugate. It is noted that polypeptides can be purifiedprior to being used to form a conjugate. Any method can be used topurify polypeptides including, without limitation, fractionation,centrifugation, and chromatography. For example, polypeptides containinga polyhistidine sequence can be purified using affinity chromatography.Once obtained, the polypeptides can be connected using any method. Forexample, a polypeptide sample can be incubated with a linking moleculesuch that individual polypeptides form conjugates. A linking molecule isany molecule that connects two polypeptides. Typically, a linkingmolecule is a molecule with two reactive groups or sites that arecapable of interacting with and thereby forming a link between aminoacid residues from two polypeptides. A linking molecule can be aspecific linking molecule such as an antibody or a non-specific linkingmolecule such as a chemical reagent (e.g., glutaraldehyde andformaldehyde).

[0038] Any antibody can be used as a linking molecule. For example, ananti-polyhistidine antibody or an anti-epitope tag antibody such as ananti-FLAG® epitope antibody or anti-hemagglutinin (HA) tag antibody canbe used to connect two polypeptides. FLAG® epitopes are described inU.S. Pat. Nos. 4,703,004 and 4,782,137. It is noted that thepolypeptides to be connected with a specific linking molecule need tocontain the specific site recognized by the linking molecule. Forexample, to connect two polypeptides with an anti-polyhistidineantibody, each polypeptide must contain the polyhistidine epitoperecognized by that antibody. For the purpose of this invention, thespecific site recognized by a specific linking molecule such as anantibody is referred to as a linker site. Any method can be used to makea polypeptide that contains a linker site such that a particularantibody can be used as a linking molecule. For example, commonmolecular cloning techniques can be used to introduce the nucleic acidthat encodes a FLAG tag epitope into the nucleic acid that encodes aparticular polypeptide. It is noted that a linker site can be located atany position. For example, a polyhistidine sequence can be at theN-terminus, C-terminus, or an internal position of a polypeptide. Inaddition, a polypeptide can contain more than one linker site. Forexample, a polypeptide can have a polyhistidine sequence at an internalposition as well as at the C-terminus. Further, a polypeptide cancontain different linker sites. For example, a polypeptide can have apolyhistidine sequence at an internal position and a FLAG tag epitope atthe C-terminus.

[0039] In some cases, two or more polypeptides can be made such thatthey are connected via a covalent bond. For example, two polypeptidescan be made as a fusion protein such that they are connected by apeptide bond. Alternatively, a polypeptide can be made in a cell linethat promotes the formation of disulfide bonds between, for example, twoidentical polypeptides. In this case, the conjugate would be ahomodimer. It is noted that any polypeptide can be engineered to containone or more cysteine residues such that the polypeptides form conjugatesvia cysteine bridges. For example, a polypeptide can be made to containN- and C-terminal cysteine residues such that conjugates of varying sizeare formed intracellularly.

[0040] In addition, the interaction between biotin and avidin can beused to form conjugates. For example, polypeptides can be designed, orchemically treated, to contain biotin molecules at the C- and N-terminalends. These biotin-containing polypeptide can be incubated with avidinmolecules that are capable of simultaneously interacting with two ormore biotin molecules. In this case, a single avidin molecule can linktwo biotin-containing polypeptides to form a conjugate. Further,chelating molecules that can simultaneously bind two or more ions (e.g.,Ni⁺⁺, Cu⁺⁺, Co⁺⁺, and Zn⁺⁺) can be used to form conjugates. For example,a copper chelating molecule that can interact with two copper ions canbe used to link two polypeptides containing a polyhistidine sequence. Inthis case, a single copper ion can interact with each polyhistidinesequence while a single copper chelating molecule links the twopolypeptides to form a conjugate. It is noted that immunostimulatingcomplexes (iscoms) can be used to form conjugates. For example, an iscomcan be designed to contain copper ions such that polypeptides containinga polyhistidine sequence can be conjugated.

[0041] Typically, a nucleic acid molecule is constructed such that aparticular polypeptide is expressed. For example, a nucleic acidmolecule can be constructed to encode a polypeptide having three similaramino acid segments as well as a polyhistidine sequence at itsC-terminus. Once constructed, the nucleic acid molecule can beintroduced into a host cell such that the polypeptide is produced. Anyhost cell can be used including, without limitation, prokaryotic cells(e.g., bacteria) and eukaryotic cells (e.g., human cells). Onceproduced, the polypeptide can be purified and used to make the desiredvaccine conjugate.

[0042] The term “nucleic acid” as used herein encompasses both RNA andDNA, including cDNA, genomic DNA, and synthetic (e.g., chemicallysynthesized) DNA. The nucleic acid can be double-stranded orsingle-stranded. Where single-stranded, the nucleic acid can be thesense strand or the antisense strand. In addition, nucleic acid can becircular or linear.

[0043] Nucleic acid can be obtained using common molecular cloning orchemical nucleic acid synthesis procedures and techniques, includingPCR. PCR refers to a procedure or technique in which target nucleic acidis amplified in a manner similar to that described in U.S. Pat. No.4,683,195, and subsequent modifications of the procedure describedtherein. Generally, sequence information from the ends of the region ofinterest or beyond are used to design oligonucleotide primers that areidentical or similar in sequence to opposite strands of a potentialtemplate to be amplified. Using PCR, a nucleic acid sequence can beamplified from RNA or DNA. For example, a nucleic acid sequence can beisolated by PCR amplification from total cellular RNA, total genomicDNA, and cDNA as well as from bacteriophage sequences, plasmidsequences, viral sequences, and the like. When using RNA as a source oftemplate, reverse transcriptase can be used to synthesize complimentaryDNA strands.

[0044] Any method can be used to introduce, nucleic acid into a cell. Infact, many methods for introducing nucleic acid into cells, whether invivo or in vitro, are well known to those skilled in the art. Forexample, calcium phosphate precipitation, electroporation, heat shock,lipofection, microinjection, and viral-mediated nucleic acid transferare common methods for introducing nucleic acid into cells. In addition,naked DNA can be delivered directly to cells in vivo as describeelsewhere (U.S. Pat. Nos. 5,580,859 and 5,589,466 includingcontinuations thereof). Further, nucleic acid can be introduced intocells by generating transgenic animals. It is noted that transgenicanimals such as rabbits, goats, sheep, and cows can be engineered suchthat large amounts of a polypeptide are secreted into their milk.

[0045] Transgenic animals can be aquatic animals (such as fish, sharks,dolphin, and the like), farm animals (such as pigs, goats, sheep, cows,horses, rabbits, and the like), rodents (such as rats, guinea pigs, andmice), non-human primates (such as baboon, monkeys, and chimpanzees),and domestic animals (such as dogs and cats). Several techniques knownin the art can be used to introduce nucleic acid into animals to producethe founder lines of transgenic animals. Such techniques include, butare not limited to, pronuclear microinjection (U.S. Pat. No. 4,873,191);retrovirus mediated gene transfer into germ lines (Van der Putten etal., Proc. Natl Acad. Sci., USA, 82:6148-6152 (1985)); gene transfectioninto embryonic stem cells (Gossler A et al., Proc Natl Acad Sci USA83:9065-9069 (1986)); gene targeting into embryonic stem cells (Thompsonet al., Cell, 56:313-321 (1989)); nuclear transfer of somatic nuclei(Schnieke AE et al., Science 278:2130-2133 (1997)); and electroporationof embryos.

[0046] For a review of techniques that can be used to generate andassess transgenic animals, skilled artisans can consult Gordon (Intl.Rev. Cytol., 115:171-229 (1989)), and may obtain additional guidancefrom, for example: Hogan et al., “Manipulating the Mouse Embryo” ColdSpring Harbor Press, Cold Spring Harbor, N.Y. (1986); Krimpenfort etal., Bio/Technology, 9:844-847 (199 1); Palmiter et al., Cell,41:343-345 (1985); Kraemer et al., “Genetic Manipulation of the EarlyMammalian Embryo” Cold Spring Harbor Press, Cold Spring Harbor, N.Y.(1985); Hammer et al., Nature, 315:680-683 (1985); Purscel et al.,Science, 244:1281-1288 (1986); Wagner et al., U.S. Pat. No. 5,175,385;and Krimpenfort et al., U.S. Pat. No. 5,175,384.

[0047] In addition, a nucleic acid that encodes a polypeptide can bemaintained within a cell in any form. For example, nucleic acid can beintegrated into the genome of a cell or maintained in an episomal state.In other words, a cell can be a stable or transient transformant.

[0048] Further, any method can be used to direct the expression of aparticular polypeptide. Such methods include, without limitation,constructing a nucleic acid such that a regulatory element promotes theexpression of a nucleic acid sequence that encodes a polypeptide.Typically, regulatory elements are DNA sequences that regulate theexpression of other DNA sequences at the level of transcription. Thus,regulatory elements include, without limitation, promoters, enhancers,and the like.

[0049] In one embodiment, a conjugate to vaccinate rats can be designedto contain polypeptides having an N-terminal polyhistidine sequencefollowed by an opossum IgE CH2 domain, a rat IgE CH3 domain, an opossumIgE CH2 domain, a rat IgE CH3 domain, an opossum IgE CH4 domain, and aC-terminal polyhistidine sequence. Alternatively, the first opossum IgECH2 domain can be followed by three rat IgE CH3 domains as opposed toonly one rat IgE CH3 domain. In either case, two polypeptides can beconnected via disulfide bonds such that dimers are formed. It is notedthat affinity chromatography can be used to purify polypeptidescontaining a polyhistidine sequence. In addition, an anti-polyhistidineantibody can be used as a linking molecule to connect any number ofsingle polypeptides or dimers through the N-terminal and C-terminalpolyhistidine sequences. For example, three dimers can be linkedsequentially via two anti-polyhistidine antibodies (i.e., dimer oneconnected to dimer two by antibody one, and dimer two connected to dimerthree by antibody two). It is noted that mixing polypeptides with alinking molecule can result in a vaccine that contains vaccineconjugates with various sizes as well as various combinations ofpolypeptides. For example, a vaccine can contain a substantial amount ofvaccine conjugates having less than four polypeptides with few havinggreater than four polypeptides. It is also noted that the generalconfiguration of the polypeptides within a vaccine conjugate can beadapted to vaccinate mammals other than rats. For example, the rat IgEdomains can be replaced with human IgE domains to vaccinate humans.

[0050] 2. Vaccine Conjugates and Cytokines

[0051] The invention provides vaccine conjugates that contain apolypeptide having a cytokine activity such that a potent immuneresponse is induced against another polypeptide within the conjugate.Such immune responses can be more potent than the responses induced by aconjugate lacking a polypeptide having a cytokine activity. Although notlimited to any particular mode of action, a vaccine conjugate containingpolypeptide X and a polypeptide having a cytokine activity presumablyconcentrates cytokine activity to the localized area containingpolypeptide X. Thus a vaccine conjugate containing a polypeptide havingcytokine activity can stimulate cells that participate in generating aspecific immune response against other polypeptides within a vaccineconjugate.

[0052] A polypeptide having cytokine activity can have any type ofcytokine activity. For example, a polypeptide can have interferon-α,interferon-β, interferon-γ, TNF-α, IL-1, IL-2, IL-4, IL-6, IL-12, IL-15,IL-18, or granulocyte-macrophage colony stimulating factor (GM-CSF)activity. It is important to note that a polypeptide having cytokineactivity can be a polypeptide that is either naturally occurring ornon-naturally occurring. A naturally occurring polypeptide is anypolypeptide having an amino acid sequence as found in nature, includingwild-type and polymorphic polypeptides. Such naturally occurringpolypeptides can be obtained from any species including, withoutlimitation, human, chimpanzee, baboon, rat, or mouse. For example, humaninterferon-α can be used in a vaccine conjugate. A non-naturallyoccurring polypeptide is any polypeptide having an amino acid sequencethat is not found in nature. Thus, a non-naturally occurring polypeptidecan be a mutated version of a naturally occurring polypeptide, or anengineered polypeptide. For example, a non-naturally occurringpolypeptide having interferon-α activity can be a mutated version of anaturally occurring polypeptide having interferon-α activity thatretains at least some interferon-α activity. A polypeptide can bemutated by, for example, sequence additions, deletions, and/orsubstitutions using standard methods such as site-directed mutagenesisof the corresponding nucleic acid coding sequence.

[0053] A conjugate can contain any number of polypeptides havingcytokine activity. For example, a conjugate can contain two polypeptideshaving cytokine activity. In addition, a conjugate can containpolypeptides having different cytokine activities. For example, aconjugate can contain one polypeptide having interferon-α activity andanother having GM-CSF activity. It is noted that polypeptides havingcytokine activity can be obtained using any method. For example, apolypeptide having cytokine activity can be designed to contain apolyhistidine sequence such that affinity chromatography can be used topurify the polypeptide. In addition, any method can be used to form aconjugate. For example, a polypeptide having cytokine activity can bedesigned to contain a linker site such that a linking molecule can linkthat polypeptide to another polypeptide such as any of the polypeptidesdescribed herein.

[0054] In one embodiment, a conjugate to vaccinate rats can be designedto contain polypeptides having cytokine activity as well as polypeptideshaving an N-terminal polyhistidine sequence followed by an opossum IgECH2 domain, a rat IgE CH3 domain, an opossum IgE CH2 domain, a rat IgECH3 domain, an opossum IgE CH4 domain, and a C-terminal polyhistidinesequence. In this case, the polypeptides having cytokine activity cancontain an N-terminal polyhistidine sequence such that affinitychromatography can be used for purification. In addition, ananti-polyhistidine antibody can be used as a linking molecule to connectany number of polypeptides via the polyhistidine sequences. For example,a conjugate can contain an interferon-α polypeptide followed by threepolypeptides containing IgE domains followed be an interferon-βpolypeptide with each connection being via an anti-polyhistidineantibody. It is noted that mixing polypeptides with a linking moleculecan result in a vaccine that contains vaccine conjugates with varioussizes and various combinations of polypeptides. For example, a vaccinecan contain a substantial amount of vaccine conjugates havingpolypeptides with interferon-α activity with few having bothpolypeptides with interferon-α activity and polypeptides withinterferon-β activity. It is also noted that the general configurationof the polypeptides within a vaccine conjugate can be adapted tovaccinate mammals other than rats. For example, the rat IgE domains canbe replaced with human IgE domains to vaccinate humans.

[0055] 3. Immunogenic Polypeptides and IgE Vaccines

[0056] For a successful IgE vaccination, it is essential to obtain astrong immune response that reacts predominantly with native IgEmolecules (e.g., IgE surface epitopes). This is required in order toachieve efficient competition with the IgE receptor for free IgE, as theinteraction between an IgE antibody and its specific IgE receptor isvery strong (2.6×10⁻¹⁰; Froese A, CRC Crit. Rev. Immunol. 1:79-132(1980)). As described herein, high levels of antibodies havingspecificity for self IgE antibodies were produced in rat strains byadministering an immunogenic polypeptide. Several different rat strainswere used including low, medium, and high IgE responders.

[0057] An immunogenic polypeptide, as described herein, is a polypeptidethat effectively induces an immune response in a mammal. For example, animmunogenic polypeptide can be a polypeptide that effectively induces ananti-self IgE response in a mammal. Typically, immunogenic polypeptidescontain at least one amino acid sequence (e.g., a single amino acidsubstitution) that would be considered non-self to a particular mammal.For example, immunogenic polypeptides that induce anti-self IgEresponses can contain two components: a self IgE portion and a non-selfIgE portion. The self IgE portion can be responsible for conferring thespecificity of the anti-self IgE response and the non-self IgE portioncan serve to promote and stabilize the immunogenic polypeptide such thatthe specific anti-self IgE response is induced. Typically, the self IgEportion of the immunogenic polypeptide is a portion of an IgE antibodythat either directly interacts with an IgE receptor or indirectlyinfluences the interaction of an IgE antibody with an IgE receptor.

[0058] Briefly, the binding site for human IgE to the high affinity IgEreceptor on mast cells and basophils is not located at the junctionbetween the CH2 and CH3 domains of IgE as previously suggested, butinstead is located in the N-terminal region of the CH3 domain. Thisregion is, due to folding, located in the junction between the CH3 andCH4 domains of the native polypeptide. Thus, use of the entire CH2-CH3domain as a self IgE portion may potentially induce an anti-self IgEresponse with antibodies interacting with self IgE antibodies alreadybound to the surface of mast cells such that anaphylactic reactionsoccur. To reduce the risk of inducing an anaphylactic response, the selfIgE portion of an immunogenic polypeptide can be the entire CH3 domainwithout the CH2 domain. Alternatively, the self IgE portion can be theN-terminal region of the CH3 domain. For example, when vaccinating arat, the self IgE portion can be the N-terminal half of the rat CH3domain in a context of a non-self IgE portion containing the entire CH2domain of opossum IgE, the C-terminal half of the CH domain of opossumIgE, and the entire CH4 domain of opossum IgE. Such an immunogenicpolypeptide can be designated ORO-trunc (FIG. 2).

[0059] Typically, the non-self IgE portion of an immunogenic polypeptidestabilizes a functional conformation of the self IgE portion. Forexample, if the self IgE portion is a CH3 domain, then the non-self IgEportion could be a CH2 domain, a CH4 domain, or a CH2 and CH4 domainwith the self CH3 domain being between the CH2 and CH4 domains.Specifically, when vaccinating a rat, the self IgE portion can be therat CH3 domain in a context of a non-self IgE portion from, for example,opossum. In this case, the rat CH3 domain can be located between theopossum CH2 and CH4 domains. Such an immunogenic polypeptide can bedesignated ORO (FIG. 2). Likewise, when vaccinating a mouse, the selfIgE portion can be the mouse CH3 domain in a context of a non-self IgEportion from, for example, opossum. Such an immunogenic polypeptide canbe designated OMO (FIG. 2).

[0060] Immunogenic polypeptides of the invention can be produced using aeukaryotic expression system, such as a mammalian cell expressionsystem. In such cases, the immunogenic polypeptide is soluble, properlyfolded, and properly modified such that an anti-self IgE response isinduced upon administration to a mammal. For example, immunogenicpolypeptides having one or more eukaryotic post-translationalmodifications can produce an anti-self IgE response that issignificantly higher than similar polypeptides lacking eukaryoticpost-translational modification (e.g., a bacterially producedpolypeptide). Eukaryotic post-translational modifications include,without limitation, glycosylation, acylation, limited proteolysis,phosphorylation, and isoprenylation. Further, soluble, properly folded,and properly modified immunogenic polypeptides can induce a stronganti-self IgE response in mammals with high concentrations of plasmaIgE, so called high IgE responders. Bacterially produced polypeptides,however, are unable to produce such a strong anti-self IgE response inhigh IgE responders. Thus, immunogenic polypeptides having highsolubility, proper folding, and proper modification can be obtained andused as described herein to induce effective anti-self IgE responses inmammals. Moreover, the immunogenic polypeptides described herein can beused to treat mammals, including humans, that have high serumconcentrations of IgE. It is noted that a high percentage of theseverely allergic patients in the human population belong to thiscategory of patients.

[0061] The IgE CH3 domain, or a portion of an IgE CH3 domain, derivedfrom an organism to be vaccinated such as human can be inserted into thestructural context of a distantly related IgE molecule such as an IgEmolecule from a non-placental mammal (e.g., opossum, platypus, koala,kangaroo, wallaby, and wombat). IgE antibodies from the grey shorttailed opossum, a marsupial, exhibit about 25 percent sequence identitywith human, rat, pig, and dog IgE antibodies. Thus, regions of theopossum IgE antibody can be used as the non-self IgE portion of animmunogenic polypeptide such that a potent anti-self IgE response isinduced in a human, rat, pig, or dog.

[0062] A nucleic acid molecule for expressing an immunogenic polypeptidecan be produced by splicing a first nucleic acid that encodes a portionof an IgE antibody from an organism to be vaccinated into a secondnucleic acid that encodes a portion of an IgE antibody from a mammaldistantly related to the organism to be vaccinated. For example, anucleic acid molecule encoding an immunogenic polypeptide containing theCH3 domain of rat, human, pig, or dog IgE can be spliced into a nucleicacid containing the CH2 and CH4 domains of opossum IgE. Such chimericnucleic acid molecules can be constructed using common molecular cloningtechniques. In general, constructing nucleic acid such that the CH3domain of an IgE antibody from one organism is positioned between theCH2 and CH4 domains of an IgE antibody from another organism results ina nucleic acid molecule that encodes a chimeric IgE molecule that hasthe CH3 domain in a structural context very similar to its nativeposition within native IgE antibodies.

[0063] When vaccinating rat, human, dog, or pig, the opossum CH2 and CH4domains can serve as the non-self IgE portion of the immunogenicpolypeptide, since there is about 30 percent amino acid identity betweenopossum CH2 and CH4 domains and the corresponding domains of rat, human,dog, and pig IgE (FIG. 1). Such immunogenic polypeptides can be producedin a mammalian host. In addition, the resulting immunogenic polypeptidescan be secreted from the mammalian producer cells in a properly foldedand properly glycosylated form. For example, analysis, in the Biacoresystem. with monoclonal antibodies directed against the CH3 domain ofhuman IgE revealed that these monoclonal antibodies can bind strongly toimmunogenic polypeptides of the invention, indicating that the entireCH3 domain can be properly folded.

[0064] It is important to note that immunogenic polypeptides describedherein can be such that deleterious side-effects are not exhibited, evenin mammals that have highly elevated IgE titres prior to vaccination. Inaddition, vaccination with an immunogenic polypeptide as describedherein can induce an anti-self IgE response that is directed against theentire free pool of IgE. Such a response is not limited to a specificallergen. Thus, these methods and materials can be used to treat humanallergies having a large variety of different atopic allergies.

[0065] 4. Additional Components and Modes of Administration

[0066] The vaccines, vaccine conjugates, and immunogenic polypeptidesdescribed herein can be administered alone or in combination with othercomponents. For example, a vaccine conjugate can contain a blockingmolecule that inhibits the interaction between an antibody (e.g., an IgGantibody) and an Fc-gamma receptor II (e.g., CD32). Such blockingmolecules (i.e., Fc-gamma receptor II blocking molecules) can include,without limitation, anti-CD32 antibodies. Anti-CD32 antibodies can beobtained using common antibody production and screening techniques. Itis noted that Fc-gamma receptor II blocking molecules can be used incombination with any immunogenic polypeptide such that the immuneresponse against the immunogenic polypeptide is enhanced. For example, amixture containing an anti-CD32 antibody and an immunogenic polypeptideeither conjugated or not can be administered to a mammal to induce apotent immune response against the immunogenic polypeptide.

[0067] To vaccinate a mammal, an effective amount of any vaccine,vaccine conjugate, or immunogenic polypeptide described herein can beadministered to a host. An effective amount refers to any amount thatinduces a desired immune response while not inducing significanttoxicity to the host. Such an amount can be determined by assessing ahost's immune response after administration of a fixed amount of aparticular material (e.g., immunization polypeptide). In addition, thelevel of toxicity, if any, can be determined by assessing a host'sclinical symptoms before and after administering a fixed amount of aparticular material. It is noted that the effective amount of aparticular material administered to a host can be adjusted according todesired outcomes as well as the host's response and level of toxicity.Significant toxicity can vary for each particular host and depends onmultiple factors including, without limitation, the host's diseasestate, age, and tolerance to pain.

[0068] In addition, any of the materials described herein can beadministered to any part of the host's body including, withoutlimitation, the joints, blood stream, lungs, intestines, muscle tissues,skin, and peritoneal cavity. Thus, a vaccine conjugate can beadministered by intravenous, intraperitoneal, intramuscular,subcutaneous, intrathecal, and intradermal injection, by oraladministration, by inhalation, or by gradual perfusion over time. Forexample, an aerosol preparation containing an immunogenic polypeptidecan be given to a host by inhalation. It is noted that the duration ofvaccination with any of the materials described herein can be any lengthof time from as short as one day to as long as a lifetime (e.g., manyyears). For example, an immunogenic polypeptide can be administered oncea year over a period of ten years. It is also noted that the frequencyof treatment can be variable. For example, an immunogenic polypeptidecan be administered once (or twice, three times, etc.) daily, weekly,monthly, or yearly.

[0069] Preparations for administration can include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents include, without limitation, propylene glycol,polyethylene glycol, vegetable oils, and injectable organic esters.Aqueous carriers include, without limitation, water as well as alcohol,saline, and buffered solutions. Preservatives, flavorings, and otheradditives such as, for example, antimicrobials, anti-oxidants, chelatingagents, inert gases, and the like may also be present. It will beappreciated that any material described herein that is to beadministered to a mammal can contain one or more commonly knownpharmaceutically acceptable carriers.

[0070] Any method can be used to determine if a particular immuneresponse is induced. For example, antibody responses against particularantigens can be determined using immunological assays (e.g., ELISA). Inaddition, clinical methods that can assess the degree of a particulardisease state can be used to determine if a desired immune response isinduced.

[0071] The invention will be further described in the followingexamples, which do not limit the scope of the invention described in theclaims.

EXAMPLES Example 1 IMMUNOGENIC POLYPEPTIDES

[0072] Nucleic acid molecules were constructed to encode immunogenicpolypeptides containing both self and non-self IgE portions. Thesenucleic acid molecules were then used to synthesize soluble immunogenicpolypeptides in mammalian cells. Such immunogenic polypeptideseffectively induced a polyclonal anti-self IgE response uponadministration to a mammal. In addition, the immunogenic polypeptidesappear to be folded and glycosylated in a manner that enabled theimmunogenic polypeptides to produce a strong and specific anti-self IgEresponse that was more potent than bacterially produced polypeptideslacking the non-self IgE portion. Thus, the immunogenic polypeptidesdescribed herein contain a majority of the surface epitopes in the sameconformation as in native plasma IgE. Moreover, immunogenic polypeptidescontaining a self IgE portion limited to either the entire CH3 domain ora fragment of the CH3 domain (e.g., N-terminal region of CH3) reducedthe potential of producing anaphylactic antibodies within a mammal.

Example 2 PRODUCTION AMD PURIFICATION OF AN IMMUNOGENIC POLYPEPTIDES

[0073] A ˜330 base pair PCR fragment encoding the CH3 domain of rat IgE(Hellman L et al., Nucleic Acids Res.10:6041-6049 (1982)) was fused withtwo similar sized fragments encoding the CH2 and CH4 domains of opossumIgE (Aveskogh M and Hellman L, Eur. J Immunol., 28:2738-2750 (1998)) byligation into a modified version of the pCEP4 expression vector,pCEP-Pu2 (Margolskee R F et al., Mol. Cell Biol. 8:2837-2847 (1988)).This vector contains the CMV promoter-enhancer, located directly 5′ ofthe coding region of interest and allows high levels of expression inmammalian cells. This vector also contains the coding regions forpuromycin resistance and the EBV EBNA1 gene. The EBNA1 gene confersmaintenance of stable replicating-episomal copies of the vector in humanor canine cell lines.

[0074] The nucleic acid molecule containing the opossum IgE CH2, rat IgECH3, and opossum IgE CH4 nucleic acid sequences also contained nucleicacid sequences that encode a signal sequence and six histidine residuesat the N-terminal region. The region containing the signal sequence andsix histidine residues facilitates secretion of the encoded polypeptidefrom producer cells and enables polypeptide purification withNi⁺⁺-chelating columns. Following transfection of the expression vectorinto human 293 cells, the opossum CH2-IgE/rat CH3-IgE/opossum CH4-IgE(ORO) immunogenic polypeptide was purified from 293 cell conditionedmedia on a nickel-chelating column to about 100 percent purity.Following elution of the ORO immunogenic polypeptide with a solutioncontaining 20 mM Tris (pH 8.0), 0.1 M NaCl, and 100 mM imidazole, theeluate was dialyzed against PBS (pH 7.5) overnight at 4° C. The OROimmunogenic polypeptide was then concentrated to about 2 mg/mL using anAmicon concentrator. An aliquot of this preparation containing the OROimmunogenic polypeptide was separated on SDS-PAGE and found to be about100 percent pure. This purified ORO immunogenic polypeptide preparationwas used as the active component of an anti-self IgE vaccine fortreating rats.

Example 3 SENSITIZATION PROCEDURE

[0075] Each rat was sensitized to ovalbumin as follows. Ten (10) μg ofovalbumin in PBS was administered to each rat intraperitoneally. Threeweeks after this initial intraperitoneal injection of ovalbumin, therats received weekly intraperitoneal injections of 1 μg of ovalbumin forfour weeks. During this four week period, the rats became sensitized toovalbumin obtaining a total IgE and ovalbumin-specific IgE response thatwas high and persistent. After this four week-period, the rats started avaccination program. During the entire vaccination program,intraperitoneal injections of ovalbumin continued as follows. During thefirst two weeks of vaccination, the rats received intraperitonealinjections of 1 μg of ovalbumin weekly. After the first two weeks ofvaccination, the rats received intraperitoneal injections of 1 μg ofovalbumin every other week.

Example 4 ELISA MEASUREMENT OF AN ANTI-SELF IgE RESPONSE

[0076] Thirty-six rats (twelve Lewis rats, twelve Louvain rats, andtwelve Brown Norway rats) were divided into two equally sized groups andinjected intraperitoneally with either the ORO immunogenic polypeptideor BSA as negative control. The BSA negative control was used at thesame polypeptide concentration as that of the ORO immunogenicpolypeptide. In this study, each rat received intraperitoneal injectionsof about 250 μg of antigen (either the ORO immunogenic polypeptide orBSA) dispersed in 0.2 mL of a 50:50 solution of Freund's completeadjuvant and PBS. Three weeks later, the rats were given a boosterinjection containing about 100 μg of antigen dispersed in 0.1 mL of a50:50 solution of Freund's incomplete adjuvant and PBS. Six weeks later,the rats were given an additional booster identical to the firstbooster. One week after this third immunization, blood samples weretaken and measured in an ELISA as follows.

[0077] The level of IgG anti-IgE antibodies directed against self ratIgE was measured by an ELISA. Native rat IgE was used at a concentrationof 5 μg/mL for coating the ELISA plates. Successive 1/5 dilutions ofserum from each of the individual rats were tested by color reaction inthe ELISA. The presence of rat IgG antibodies having specificity for ratIgE antibodies was determined using two biotinylated mouse monoclonalantibodies, one with specificity for rat IgG2a/b and one for rat IgG1.Alkaline phosphatase coupled strepavidin was used to detect thesebiotinylated mouse monoclonal antibodies.

Example 5 INDUCTION OF AN ANTI-SELF IgE RESPONSE IN A MAMMAL

[0078] The in vivo effect of the ORO immunogenic polypeptide as an IgEvaccine was investigated using three different strains of rats (Lewis,Louvain, and Brown Norway). Lewis rats are low IgE responders, Louvainrats are medium IgE responders, and Brown Norway rats are high IgEresponders. After sensitization to ovalbumin, each rat was vaccinatedwith either the ORO immunogenic polypeptide or BSA as described inExample 3. After collecting blood samples, the sera was diluted in stepsof five as indicated (FIG. 3). Purified monoclonal rat IgE (IR 162) wasused to coat the ELISA plates (5 μg/mL) and two biotinylated mousemonoclonal antibodies were used to detect rat IgG anti-IgE antibodies.Following the second booster dose, high anti-IgE titres were detected inthe low, medium, and high IgE responding rats that received the vaccinecontaining the ORO immunogenic polypeptide. Anti-IgE titers were notdetected in rats treated with the BSA control. Thus, the ORO immunogenicpolypeptide was capable of inducing an anti-self IgE response in ratsthat contained low, medium, and high amounts of IgE antibodies.

[0079] A difference in anti-self IgE levels between the various strainswas observed. The low responder strain, Lewis, showed very highanti-self IgE titres. The sera could be diluted more than 3000 timesbefore a significant decrease in OD values upon ELISA measurements wasdetected (FIG. 3). For the high responder strain, Brown Norway, however,the OD values started to drop for three out of six animals at a dilutionof 25 times or more (FIG. 3).

[0080] In another experiment, Wistar rats were used. Wistar rats aremedium IgE responders. The anti-self IgE response produced by the Wistarrats was similar to the response observed in the Lewis rats.

Example 6 ANALYSIS OF CROSS REACTIVITY

[0081] The cross reactivity between rat antibodies directed against anopossum IgE CH2 or CH4 domain with the corresponding domain of rat IgEantibodies was evaluated. This potential cross reactivity could resultfrom a low primary amino acid sequence homology or a close structuralsimilarity between the CH2 and CH4 domains of opossum IgE and rat IgE.The induction of an anti-rat IgE response having specificity for the ratCH2 or CH4 domains could lead to mast cell activation.

[0082] A recombinant polypeptide (OOO) containing the opossumCH2-CH3-CH4 domains was injected into the Wistar strain. After a secondbooster injection, sera from these rats were collected and tested forthe presence of antibodies having specificity for rat IgE. No anti-ratIgE antibodies were detected in the rats treated with the OOOpolypeptide (FIG. 4). In addition, Wistar rats treated with the OROimmunogenic polypeptide exhibited an anti-self IgE response similar tothat observed in Lewis rats. Further, Wistar rats treated with arecombinant polypeptide (PPP) containing platypus CH2-CH3-CH4 domainsdid not produce an anti-rat IgE response. Thus, the CH2, CH3, and CH4domains of opossum and platypus IgE antibodies do not generate, uponadministration to rats, rat antibodies having specificity for rat IgEantibodies.

[0083] The interaction between the rat antibodies induced by the OROimmunogenic polypeptide (rat IgG anti-self IgE antibodies) and human IgEantibodies was examined. In a few cases, minor cross reactivity wasobserved. This minor cross reactivity detected in a few rats was mostlikely caused by the interaction between rat IgG anti-rat CH3 IgEantibodies and the CH3 domain of human IgE. Since the CH3 domains of ratand human IgE are much more closely related than human and opossum orplatypus IgE, vaccines containing opossum or platypus components can beconsidered highly safe, presenting minimal risk for the generation ofcross linking antibodies.

Example 7 POLYPEPTIDES FOR VACCINE CONJUGATES

[0084] The nucleic acid construct encoding the ORO immunogenicpolypeptide described in Example 2 was used to produce two polypeptideseach having several identical self epitopes. One polypeptide containedtwo identical clusters of self epitopes (the entire rat CH3 domain),while the other polypeptide contained four such clusters. First, nucleicacid encoding six histidine residues was added to the C-terminal end ofthe opossum CH4 domain by including a nucleotide sequence for sixhistidine residues in the 3′ PCR primer. Thus, each polypeptidecontained a polyhistidine sequence at both the N- and C-terminal ends sothat conjugates can be formed. Second, a nucleic acid fragment encodingthe opossum CH2 and the rat CH3 domains of the original construct wasobtained by PCR amplification. This fragment was subsequently ligatedinto the construct encoding the ORO immunogenic polypeptide. Theresulting construct encoded a polypeptide designated ORORO. This OROROpolypeptide contains two rat CH3 domains, two opossum CH2 domains, andone opossum CH4 domain in the following order opossum CH2, rat CH3,opossum CH2, rat CH3, and opossum CH4. Thus, this polypeptide has twoidentical CH3 domains, each with multiple self epitopes.

[0085] The nucleic acid construct encoding ORORO was used as startingmaterial to produce the second immunogenic polypeptide. This polypeptidecontains two additional rat CH3 domains that were added to a position 3′of the first rat CH3 domain in the ORORO polypeptide. The resultingpolypeptide has a polyhistidine tag followed by an opossum CH2 domain,three identical rat CH3 domains, one opossum CH2 domain, one rat CH3domain, an opossum CH4 domain, and a C-terminal polyhistidine tag(6his-ORRRORO-6his).

[0086] Each recombinant polypeptide was produced in the pCEP4 basedvector system. In addition, the polypeptides were purified usingNi⁺⁺chelating columns according to the method described in Example 2.Similar vaccine constructs are produced using dog or human IgE CH3domain instead of the rat IgE CH3 domain.

Example 8 VACCINE CONJUGATES

[0087] To determine a favorable combination of polypeptide to monoclonalantibody, the purified polypeptides of Example 7 are mixed with amonoclonal anti-polyhistidine antibody in various combinations rangingfrom a 1/1 to a 10/1 ratio (polypeptide to monoclonal antibody ratio).This mixture results in the generation of long multimeric conjugateswith a large number of identical self epitopes in tandem. The biologicalactivity of the various combinations is assessed in rats as describedherein. The non-conjugated ORO immunogenic polypeptide is used as areference to assess immune responses.

Example 9 POLYPEPTIDES HAVING CYTOKINE ACTIVITY

[0088] PCR primers are designed so that cDNAs encoding rat, dog, andhuman cytokines (e.g., interferon-α, interferon-γ, and GM-CSF) can beisolated from total spleen mRNA. The nucleotide sequence encoding sixhistidine residues is introduced into each 5′ PCR primer so that thecytokines can be purified via affinity chromatography and can benon-covalently conjugated to the polypeptides described herein via ananti-polyhistidine antibody. The recombinant cytokines are producedusing any one of the following three expression systems: bacteria, yeast(e.g., Pichia pastoris), and mammalian cells (e.g., 293-EBNA cells usinga pCEP-4 based expression system).

Example 10 VACCINE CONJUGATES HAVING A POLYPEPTIDE WITH CYTOKINEACTIVITY

[0089] Cytokines produced according to Example 9 are used to makevaccine conjugates. A mixture of three different cytokines (e.g., mouseinterferon-α, rat interferon-γ and rat GM-CSF) is produced and tested incombination with the ORORO polypeptide and an anti-polyhistidineantibody.

[0090] Initially, a mixture of 1/10 ratio (cytokine to immunogenicpolypeptide ratio) is tested. With three cytokines, this ratio resultsin a mixture of 30% cytokine per molar basis and 70% immunogenicpolypeptide. In addition, this mixture contains an anti-polyhistidineantibody at a 1/10 ratio (immunogenic polypeptide to monoclonal antibodyratio). A large number of ratio combinations is evaluated so that anoptimal cytokine to immunogenic polypeptide ratio as well as an optimalmonoclonal antibody to immunogenic polypeptide ratio is determined. Inaddition, polypeptides having cytokine activity from various species isassessed to determine the optimal combination for a particular species.

OTHER EMBODIMENTS

[0091] It is to be understood that while the invention has beendescribed in conjunction with the detailed description thereof, theforegoing description is intended to illustrate and not limit the scopeof the invention, which is defined by the scope of the appended claims.Other aspects, advantages, and modifications are within the scope of thefollowing claims.

1 11 1 331 PRT Artificial Sequence Synthetically generated proteins 1Asp Asn Lys Thr Phe Ser Val Cys Ser Arg Asp Phe Thr Pro Pro Thr 1 5 1015 Val Lys Ile Leu Gln Ser Ser Cys Asp Gly Gly Gly His Phe Pro Pro 20 2530 Thr Ile Gln Leu Leu Cys Leu Val Ser Gly Tyr Thr Pro Gly Thr Ile 35 4045 Asn Ile Thr Trp Leu Glu Asp Gly Gln Val Met Asp Val Asp Leu Ser 50 5560 Thr Ala Ser Thr Thr Gln Glu Gly Glu Leu Ala Ser Thr Gln Ser Glu 65 7075 80 Leu Thr Leu Ser Gln Lys His Trp Leu Ser Asp Arg Thr Tyr Thr Cys 8590 95 Gln Val Thr Tyr Gln Gly His Thr Phe Glu Asp Ser Thr Lys Lys Cys100 105 110 Ala Asp Ser Asn Pro Arg Gly Val Ser Ala Tyr Leu Ser Arg ProSer 115 120 125 Pro Phe Asp Leu Phe Ile Arg Lys Ser Pro Thr Ile Thr CysLeu Val 130 135 140 Val Asp Leu Ala Pro Ser Lys Gly Thr Val Asn Leu ThrTrp Ser Arg 145 150 155 160 Ala Ser Gly Lys Pro Val Asn His Ser Thr ArgLys Glu Glu Lys Gln 165 170 175 Arg Asn Gly Thr Leu Thr Val Thr Ser ThrLeu Pro Val Gly Thr Arg 180 185 190 Asp Trp Ile Glu Gly Glu Thr Tyr GlnCys Arg Val Thr His Pro His 195 200 205 Leu Pro Arg Ala Leu Met Arg SerThr Thr Lys Thr Ser Gly Pro Arg 210 215 220 Ala Ala Pro Glu Val Tyr AlaPhe Ala Thr Pro Glu Trp Pro Gly Ser 225 230 235 240 Arg Asp Lys Arg ThrLeu Ala Cys Leu Ile Gln Asn Phe Met Pro Glu 245 250 255 Asp Ile Ser ValGln Trp Leu His Asn Glu Val Gln Leu Pro Asp Ala 260 265 270 Arg His SerThr Thr Gln Pro Arg Lys Thr Lys Gly Ser Gly Phe Phe 275 280 285 Val PheSer Arg Leu Glu Val Thr Arg Ala Glu Trp Glu Gln Lys Asp 290 295 300 GluPhe Ile Cys Arg Ala Val His Glu Ala Ala Ser Pro Ser Gln Thr 305 310 315320 Val Gln Arg Ala Val Ser Val Asn Pro Gly Lys 325 330 2 340 PRTArtificial Sequence Synthetically generated proteins 2 Asp Leu Thr IleArg Ala Arg Pro Val Asn Ile Thr Lys Pro Thr Val 1 5 10 15 Asp Leu LeuHis Ser Ser Cys Asp Pro Asn Ala Phe His Ser Thr Ile 20 25 30 Gln Leu TyrCys Phe Val Tyr Gly His Ile Gln Asn Asp Val Ser Ile 35 40 45 His Trp LeuMet Asp Asp Arg Lys Ile Tyr Glu Thr His Ala Gln Asn 50 55 60 Val Leu IleLys Glu Glu Gly Lys Leu Ala Ser Thr Tyr Ser Arg Leu 65 70 75 80 Asn IleThr Gln Gln Gln Trp Met Ser Glu Ser Thr Phe Thr Cys Lys 85 90 95 Val ThrSer Gln Gly Glu Asn Tyr Trp Ala His Thr Arg Arg Cys Ser 100 105 110 AspAsp Glu Pro Arg Gly Val Ile Thr Tyr Leu Ile Pro Pro Ser Pro 115 120 125Leu Asp Leu Tyr Glu Asn Gly Thr Pro Lys Leu Thr Cys Leu Val Leu 130 135140 Asp Leu Glu Ser Glu Glu Asn Ile Thr Val Thr Trp Val Arg Glu Arg 145150 155 160 Lys Lys Ser Ile Gly Ser Ala Ser Gln Arg Ser Thr Lys His HisAsn 165 170 175 Ala Thr Thr Ser Ile Thr Ser Ile Leu Pro Val Asp Ala LysAsp Trp 180 185 190 Ile Glu Gly Glu Gly Tyr Gln Cys Arg Val Asp His ProHis Phe Pro 195 200 205 Lys Pro Ile Val Arg Ser Ile Thr Lys Ala Pro GlyLys Arg Ser Ala 210 215 220 Pro Glu Val Tyr Val Phe Leu Pro Pro Glu GluGlu Glu Lys Asp Lys 225 230 235 240 Arg Thr Leu Thr Cys Leu Ile Gln AsnPhe Phe Pro Glu Asp Ile Ser 245 250 255 Val Gln Trp Leu Gln Asp Ser LysLeu Ile Pro Lys Ser Gln His Ser 260 265 270 Thr Thr Thr Pro Leu Lys TyrAsn Gly Ser Asn Gln Arg Phe Phe Ile 275 280 285 Phe Ser Arg Leu Glu ValThr Lys Ala Leu Trp Thr Gln Thr Lys Gln 290 295 300 Phe Thr Cys Arg ValIle His Glu Ala Leu Arg Glu Pro Arg Lys Leu 305 310 315 320 Glu Arg ThrIle Ser Lys Ser Leu Gly Asn Thr Ser Leu Arg Pro Ser 325 330 335 Gln AlaSer Met 340 3 341 PRT Artificial Sequence Synthetically generatedproteins 3 Glu Phe His His His His His His Thr Leu Ser Leu Pro Glu SerGly 1 5 10 15 Pro Val Thr Ile Ile Pro Pro Thr Val Lys Leu Phe His SerSer Cys 20 25 30 Asp Pro Arg Gly Asp Ala His Ser Thr Ile Gln Leu Leu CysLeu Val 35 40 45 Ser Gly Phe Ser Pro Ala Lys Val His Val Thr Trp Leu ValAsp Gly 50 55 60 Gln Glu Ala Glu Asn Leu Phe Pro Tyr Thr Thr Arg Pro LysArg Glu 65 70 75 80 Gly Gly Gln Thr Phe Ser Leu Gln Ser Glu Val Asn IleThr Gln Gly 85 90 95 Gln Trp Met Ser Ser Asn Thr Tyr Thr Cys His Val LysHis Asn Gly 100 105 110 Ser Ile Phe Glu Asp Ser Ala Gln Lys Cys Ser AspThr Asp Pro Arg 115 120 125 Gly Ile Ser Ala Tyr Ile Leu Pro Pro Thr ProGln Asp Leu Phe Val 130 135 140 Lys Lys Val Pro Thr Ile Gly Cys Leu IleVal Asp Leu Ala Ser Ala 145 150 155 160 Glu Asn Val Lys Val Thr Trp SerArg Glu Ser Gly Gly Pro Val Asn 165 170 175 Pro Ser Ser Leu Val Val LysGlu Gln Tyr Asn Gly Thr Phe Thr Val 180 185 190 Thr Ser His Leu Pro ValAsn Thr Asp Asp Trp Ile Glu Gly Asp Thr 195 200 205 Tyr Thr Cys Arg LeuGlu Ser Pro Asp Met Pro Val Pro Leu Ile Arg 210 215 220 Thr Ile Ser LysAla Pro Gly Lys Arg Leu Ala Pro Glu Val Tyr Met 225 230 235 240 Leu ProPro Ser Pro Glu Glu Thr Gly Thr Thr Arg Thr Val Thr Cys 245 250 255 LeuIle Arg Gly Phe Tyr Pro Ser Glu Ile Ser Val Gln Trp Leu Phe 260 265 270Asn Asn Glu Glu Asp His Thr Gly His His Thr Thr Thr Arg Pro Gln 275 280285 Lys Asp His Gly Thr Asp Pro Ser Phe Phe Leu Tyr Ser Arg Met Leu 290295 300 Val Asn Lys Ser Ile Trp Glu Lys Gly Asn Leu Val Thr Cys Arg Val305 310 315 320 Val His Glu Ala Leu Pro Gly Ser Arg Thr Leu Glu Lys SerLeu His 325 330 335 Tyr Ser Ala Gly Asn 340 4 341 PRT ArtificialSequence Synthetically generated proteins 4 Glu Phe His His His His HisHis Thr Leu Ser Leu Pro Glu Ser Gly 1 5 10 15 Pro Val Thr Ile Ile ProPro Thr Val Lys Leu Phe His Ser Ser Cys 20 25 30 Asp Pro Arg Gly Asp AlaHis Ser Thr Ile Gln Leu Leu Cys Leu Val 35 40 45 Ser Gly Phe Ser Pro AlaLys Val His Val Thr Trp Leu Val Asp Gly 50 55 60 Gln Glu Ala Glu Asn LeuPhe Pro Tyr Thr Thr Arg Pro Lys Arg Glu 65 70 75 80 Gly Gly Gln Thr PheSer Leu Gln Ser Glu Val Asn Ile Thr Gln Gly 85 90 95 Gln Trp Met Ser SerAsn Thr Tyr Thr Cys His Val Lys His Asn Gly 100 105 110 Ser Ile Phe GluAsp Ser Ser Arg Arg Cys Ser Asp Asp Glu Pro Arg 115 120 125 Gly Val IleThr Tyr Leu Ile Pro Pro Ser Pro Leu Asp Leu Tyr Glu 130 135 140 Asn GlyThr Pro Lys Leu Thr Cys Leu Val Leu Asp Leu Glu Ser Glu 145 150 155 160Glu Asn Ile Thr Val Thr Trp Val Arg Glu Arg Lys Lys Ser Ile Gly 165 170175 Ser Ala Ser Gln Arg Ser Thr Lys His His His Ala Thr Thr Ser Ile 180185 190 Thr Ser Ile Leu Pro Val Asp Ala Lys Asp Trp Ile Glu Gly Glu Gly195 200 205 Tyr Gln Cys Arg Val Asp His Pro His Phe Pro Lys Pro Ile ValArg 210 215 220 Ser Ile Thr Lys Leu Pro Gly Lys Arg Leu Ala Pro Glu ValTyr Met 225 230 235 240 Leu Pro Pro Ser Pro Glu Glu Thr Gly Thr Thr ArgThr Val Thr Cys 245 250 255 Leu Ile Arg Gly Phe Tyr Pro Ser Glu Ile SerVal Gln Trp Leu Pro 260 265 270 Asn Asn Glu Glu Asp His Thr Gly His HisThr Thr Thr Arg Pro Gln 275 280 285 Lys Asp His Gly Thr Asp Pro Ser PhePhe Leu Tyr Ser Arg Met Leu 290 295 300 Val Asn Lys Ser Ile Trp Glu LysGly Asn Leu Val Thr Cys Arg Val 305 310 315 320 Val His Glu Ala Leu ProGly Ser Arg Thr Leu Glu Lys Ser Leu His 325 330 335 Tyr Ser Ala Gly Asn340 5 342 PRT Artificial Sequence Synthetically generated proteins 5 GluPhe His His His His His His Thr Leu Ser Leu Pro Glu Ser Gly 1 5 10 15Pro Val Thr Ile Ile Pro Pro Thr Val Lys Leu Phe His Ser Ser Cys 20 25 30Asp Pro Arg Gly Asp Ala His Ser Thr Ile Gln Leu Leu Cys Leu Val 35 40 45Ser Gly Phe Ser Pro Ala Lys Val His Val Thr Trp Leu Val Asp Gly 50 55 60Gln Glu Ala Glu Asn Leu Phe Pro Tyr Thr Thr Arg Pro Lys Arg Glu 65 70 7580 Gly Gly Gln Thr Phe Ser Leu Gln Ser Glu Val Asn Ile Thr Gln Gly 85 9095 Gln Trp Met Ser Ser Asn Thr Tyr Thr Cys His Val Lys His Asn Gly 100105 110 Ser Ile Phe Glu Asp Ser Ser Arg Arg Cys Ser Asp Asp Glu Pro Arg115 120 125 Gly Val Ile Thr Tyr Leu Ile Pro Pro Ser Pro Leu Asp Leu TyrGlu 130 135 140 Asn Gly Thr Pro Lys Leu Thr Cys Leu Val Leu Asp Leu GluSer Glu 145 150 155 160 Glu Asn Ile Thr Val Thr Trp Val Arg Glu Arg LysLys Ser Ile Gly 165 170 175 Ser Ala Arg Ser Leu Val Val Lys Glu Gln TyrAsn Gly Thr Phe Thr 180 185 190 Val Thr Ser His Leu Pro Val Asn Thr AspAsp Trp Ile Glu Gly Asp 195 200 205 Thr Tyr Thr Cys Arg Leu Glu Ser ProAsp Met Pro Tyr Pro Leu Ile 210 215 220 Arg Thr Ile Ser Lys Ala Pro GlyLys Arg Leu Ala Pro Glu Val Tyr 225 230 235 240 Met Leu Pro Pro Ser ProGlu Glu Thr Gly Thr Thr Arg Thr Val Thr 245 250 255 Cys Leu Ile Arg GlyPhe Tyr Pro Ser Glu Ile Ser Val Gln Trp Leu 260 265 270 Pro Asn Asn GluGlu Asp His Thr Gly His His Thr Thr Thr Arg Pro 275 280 285 Gln Lys AspHis Gly Thr Asp Pro Ser Phe Phe Leu Tyr Ser Arg Met 290 295 300 Leu ValAsn Lys Ser Ile Trp Glu Lys Gly Asn Leu Val Thr Cys Arg 305 310 315 320Val Val His Glu Ala Leu Pro Gly Ser Arg Thr Leu Glu Lys Ser Leu 325 330335 His Tyr Ser Ala Gly Asn 340 6 341 PRT Artificial SequenceSynthetically generated proteins 6 Glu Phe His His His His His His ThrLeu Ser Leu Pro Glu Ser Gly 1 5 10 15 Pro Val Thr Ile Ile Pro Pro ThrVal Lys Leu Phe His Ser Ser Cys 20 25 30 Asp Pro Arg Gly Asp Ala His SerThr Ile Gln Leu Leu Cys Leu Val 35 40 45 Ser Gly Phe Ser Pro Ala Lys ValHis Val Thr Trp Leu Val Asp Gly 50 55 60 Gln Glu Ala Glu Asn Leu Phe ProTyr Thr Thr Arg Pro Lys Arg Glu 65 70 75 80 Gly Gly Gln Thr Phe Ser LeuGln Ser Glu Val Asn Ile Thr Gln Gly 85 90 95 Gln Trp Met Ser Ser Asn ThrTyr Thr Cys His Val Lys His Asn Gly 100 105 110 Ser Ile Phe Glu Asp SerSer Arg Arg Cys Pro Asp His Glu Pro Arg 115 120 125 Gly Val Ile Thr TyrLeu Ile Pro Pro Ser Pro Leu Asp Leu Tyr Gln 130 135 140 Asn Gly Ala ProLys Leu Thr Cys Leu Val Val Asp Leu Glu Ser Glu 145 150 155 160 Lys AsnVal Asn Val Thr Trp Asn Gln Glu Lys Lys Thr Ser Val Asn 165 170 175 AlaSer Gln Trp Tyr Thr Lys His His Asn Asn Ala Thr Thr Ser Ile 180 185 190Thr Ser Ile Leu Pro Val Val Ala Lys Asp Trp Ile Glu Gly Tyr Gly 195 200205 Tyr Gln Cys Ile Val Asp His Pro Asp Phe Pro Lys Pro Ile Val Arg 210215 220 Ser Ile Thr Lys Leu Pro Gly Lys Arg Leu Ala Pro Glu Val Tyr Met225 230 235 240 Leu Pro Pro Ser Pro Glu Glu Thr Gly Thr Thr Arg Thr ValThr Cys 245 250 255 Leu Ile Arg Gly Phe Tyr Pro Ser Glu Ile Ser Val GlnTrp Leu Pro 260 265 270 Asn Asn Glu Glu Asp His Thr Gly His His Thr ThrThr Arg Pro Gln 275 280 285 Lys Asp His Gly Thr Asp Pro Ser Phe Phe LeuTyr Ser Arg Met Leu 290 295 300 Val Asn Lys Ser Ile Trp Glu Lys Gly AsnLeu Val Thr Cys Arg Val 305 310 315 320 Val His Glu Ala Leu Pro Gly SerArg Thr Leu Glu Lys Ser Leu His 325 330 335 Tyr Ser Ala Gly Asn 340 7343 PRT Artificial Sequence Synthetically generated proteins 7 Glu PheHis His His His His His Thr Glu Val Tyr Ser Asp Ser Ser 1 5 10 15 LysAsp Pro Ile Pro Pro Thr Val Lys Leu Leu His Ser Ser Cys Asp 20 25 30 ProArg Gly Asp Ser Gln Ala Ser Ile Glu Leu Leu Cys Leu Ile Thr 35 40 45 GlyTyr Ser Pro Ala Gly Ile Gln Val Asp Trp Leu Val Asp Gly Gln 50 55 60 LysAla Glu Asn Leu Phe Pro Tyr Thr Ala Pro Pro Lys Arg Glu Gly 65 70 75 80Asn Arg Ser Phe Ser Ser His Ser Glu Val Asn Ile Thr Gln Asp Gln 85 90 95Trp Leu Ser Gly Lys Thr Phe Thr Cys Gln Val Thr His Leu Ala Asp 100 105110 Lys Lys Thr Tyr Gln Asp Ser Ala Pro Lys Cys Ala Asp Ser Asp Pro 115120 125 Arg Gly Ile Thr Val Phe Ile Thr Pro Pro Ser Pro Thr Asp Leu Tyr130 135 140 Ile Ser Lys Thr Pro Lys Leu Thr Cys Leu Ile Ile Asp Leu ValSer 145 150 155 160 Thr Glu Gly Met Glu Val Thr Trp Ser Arg Glu Ser GlyThr Pro Leu 165 170 175 Ser Ala Glu Ser Phe Glu Glu Gln Lys Gln Phe AsnGly Thr Met Ser 180 185 190 Phe Ile Ser Thr Val Pro Val Asn Ile Gln AspTrp Asn Arg Gly Glu 195 200 205 Ser Tyr Thr Cys Pro Val Ala His Pro AspLeu Pro Ser Pro Ile Ile 210 215 220 Lys Thr Val Thr Lys Leu Pro Gly LysPro Leu Ala Pro Glu Val Tyr 225 230 235 240 Ala Phe Pro Pro His Gln AlaGlu Val Ser His Gly Ala Ser Leu Ser 245 250 255 Leu Thr Cys Leu Ile ProGly Phe Tyr Pro Glu Asn Ile Ser Val Arg 260 265 270 Trp Leu Leu Asp AsnLys Pro Leu Pro Thr Glu His Tyr Arg Thr Thr 275 280 285 Lys Pro Leu LysAsp Gln Gly Pro Asp Pro Ala Tyr Phe Leu Tyr Ser 290 295 300 Pro Leu AlaVal Asn Lys Ser Thr Trp Glu Gln Gly Asn Val Tyr Thr 305 310 315 320 CysGln Val Val His Glu Ala Leu Pro Ser Arg Asn Thr Glu Arg Lys 325 330 335Phe Gln His Thr Ser Gly Asn 340 8 342 PRT Artificial SequenceSynthetically generated proteins 8 Glu Phe His His His His His His ThrLeu Ser Leu Pro Glu Ser Gly 1 5 10 15 Pro Val Thr Ile Ile Pro Pro ThrVal Lys Leu Phe His Ser Ser Cys 20 25 30 Asp Pro Arg Gly Asp Ala His SerThr Ile Gln Leu Leu Cys Leu Val 35 40 45 Ser Gly Phe Ser Pro Ala Lys ValHis Val Thr Trp Leu Val Asp Gly 50 55 60 Gln Glu Ala Glu Asn Leu Phe ProTyr Thr Thr Arg Pro Lys Arg Glu 65 70 75 80 Gly Gly Gln Thr Phe Ser LeuGln Ser Glu Val Asn Ile Thr Gln Gly 85 90 95 Gln Trp Met Ser Ser Asn ThrTyr Thr Cys His Val Lys His Asn Gly 100 105 110 Ser Ile Phe Glu Asp SerSer Arg Lys Cys Ala Asp Ser Asn Pro Arg 115 120 125 Gly Val Ser Ala TyrLeu Ser Arg Pro Ser Pro Phe Asp Leu Phe Ile 130 135 140 Arg Lys Ser ProThr Ile Thr Cys Leu Val Val Asp Leu Ala Pro Ser 145 150 155 160 Lys GlyThr Val Asn Leu Thr Trp Ser Arg Ala Ser Gly Lys Pro Val 165 170 175 AsnHis Ser Thr Arg Lys Glu Glu Lys Gln Arg Asn Gly Thr Leu Thr 180 185 190Val Thr Ser Thr Leu Pro Val Gly Thr Arg Asp Trp Ile Glu Gly Glu 195 200205 Thr Tyr Gln Cys Arg Val Thr His Pro His Leu Pro Arg Ala Leu Met 210215 220 Arg Ser Thr Thr Lys Leu Pro Gly Lys Arg Leu Ala Pro Glu Val Tyr225 230 235 240 Met Leu Pro Pro Ser Pro Glu Glu Thr Gly Thr Thr Arg ThrVal Thr 245 250 255 Cys Leu Ile Arg Gly Phe Tyr Pro Ser Glu Ile Ser ValGln Trp Leu 260 265 270 Phe Asn Asn Glu Glu Asp His Thr Gly His His ThrThr Thr Arg Pro 275 280 285 Gln Lys Asp His Gly Thr Asp Pro Ser Phe PheLeu Tyr Ser Arg Met 290 295 300 Leu Val Asn Lys Ser Ile Trp Glu Lys GlyAsn Leu Val Thr Cys Arg 305 310 315 320 Val Val His Glu Ala Leu Pro GlySer Arg Thr Leu Glu Lys Ser Leu 325 330 335 His Tyr Ser Ala Gly Asn 3409 341 PRT Artificial Sequence Synthetically generated proteins 9 Glu PheHis His His His His His Thr Leu Ser Leu Pro Glu Ser Gly 1 5 10 15 ProVal Thr Ile Ile Pro Pro Thr Val Lys Leu Phe His Ser Ser Cys 20 25 30 AspPro Arg Gly Asp Ala His Ser Thr Ile Gln Leu Leu Cys Leu Val 35 40 45 SerGly Phe Ser Pro Ala Lys Val His Val Thr Trp Leu Val Asp Gly 50 55 60 GlnGlu Ala Glu Asn Leu Phe Pro Tyr Thr Thr Arg Pro Lys Arg Glu 65 70 75 80Gly Gly Gln Thr Phe Ser Leu Gln Ser Glu Val Asn Ile Thr Gln Gly 85 90 95Gln Trp Met Ser Ser Asn Thr Tyr Thr Cys His Val Lys His Asn Gly 100 105110 Ser Ile Phe Glu Asp Ser Ser Arg Arg Cys Ser Asp Asp Glu Pro Arg 115120 125 Gly Val Ile Thr Tyr Leu Ile Pro Pro Ser Pro Leu Asp Leu Tyr Glu130 135 140 Asn Gly Thr Pro Lys Leu Thr Cys Leu Val Leu Asp Leu Glu SerGlu 145 150 155 160 Glu Asn Ile Thr Val Thr Trp Val Arg Glu Arg Lys LysSer Ile Gly 165 170 175 Ser Ala Ser Gln Arg Ser Thr Lys His His Asn AlaThr Thr Ser Ile 180 185 190 Thr Ser Ile Leu Pro Val Asp Ala Lys Asp TrpIle Glu Gly Glu Gly 195 200 205 Tyr Gln Cys Arg Val Asp His Pro His PhePro Lys Pro Ile Val Arg 210 215 220 Ser Ile Thr Lys Leu Pro Gly Lys ArgLeu Ala Pro Glu Val Tyr Met 225 230 235 240 Leu Pro Pro Ser Pro Glu GluThr Gly Thr Thr Arg Thr Val Thr Cys 245 250 255 Leu Ile Arg Gly Phe TyrPro Ser Glu Ile Ser Val Gln Trp Leu Phe 260 265 270 Asn Asn Glu Glu AspHis Thr Gly His His Thr Thr Thr Arg Pro Gln 275 280 285 Lys Asp His GlyThr Asp Pro Ser Phe Phe Leu Tyr Ser Arg Met Leu 290 295 300 Val Asn LysSer Ile Trp Glu Lys Gly Asn Leu Val Thr Cys Arg Val 305 310 315 320 ValHis Glu Ala Leu Pro Gly Ser Arg Thr Leu Glu Lys Ser Leu His 325 330 335Tyr Ser Ala Gly Asn 340 10 345 PRT Artificial Sequence Syntheticallygenerated proteins 10 Glu Phe His His His His His His Thr Leu Ser LeuPro Glu Ser Gly 1 5 10 15 Pro Val Thr Ile Ile Pro Pro Thr Val Lys LeuPhe His Ser Ser Cys 20 25 30 Asp Pro Arg Gly Asp Ala His Ser Thr Ile GlnLeu Leu Cys Leu Val 35 40 45 Ser Gly Phe Ser Pro Ala Lys Val His Val ThrTrp Leu Val Asp Gly 50 55 60 Gln Glu Ala Glu Asn Leu Phe Pro Tyr Thr ThrArg Pro Lys Arg Glu 65 70 75 80 Gly Gly Gln Thr Phe Ser Leu Gln Ser GluVal Asn Ile Thr Gln Gly 85 90 95 Gln Trp Met Ser Ser Asn Thr Tyr Thr CysHis Val Lys His Asn Gly 100 105 110 Ser Ile Phe Glu Asp Ser Ser Arg ArgCys Thr Ala Glu Ser Glu Pro 115 120 125 Arg Gly Val Ser Ala Tyr Leu SerPro Pro Thr Pro Leu Asp Leu Tyr 130 135 140 Val His Lys Ser Pro Lys LeuThr Cys Leu Val Val Asp Leu Ala Ser 145 150 155 160 Ser Glu Asn Val AsnLeu Leu Trp Ser Arg Glu Asn Lys Gly Gly Val 165 170 175 Ile Leu Pro ProPro Gly Pro Pro Val Ile Lys Pro Gln Phe Asn Gly 180 185 190 Thr Phe SerAla Thr Ser Thr Leu Pro Val Asn Val Ser Asp Trp Ile 195 200 205 Glu GlyGlu Thr Tyr Tyr Cys Asn Val Thr His Pro Asp Leu Pro Lys 210 215 220 ProIle Leu Arg Ser Ile Ser Lys Leu Pro Gly Lys Arg Leu Ala Pro 225 230 235240 Glu Val Tyr Met Leu Pro Pro Ser Pro Glu Glu Thr Gly Thr Thr Arg 245250 255 Thr Val Thr Cys Leu Ile Arg Gly Phe Tyr Pro Ser Glu Ile Ser Val260 265 270 Gln Trp Leu Phe Asn Asn Glu Glu Asp His Thr Gly His His ThrThr 275 280 285 Thr Arg Pro Gln Lys Asp His Gly Thr Asp Pro Ser Phe PheLeu Tyr 290 295 300 Ser Arg Met Leu Val Asn Lys Ser Ile Trp Glu Lys GlyAsn Leu Val 305 310 315 320 Thr Cys Arg Val Val His Glu Ala Leu Pro GlySer Arg Thr Leu Glu 325 330 335 Lys Ser Leu His Tyr Ser Ala Gly Asn 340345 11 341 PRT Artificial Sequence Synthetically generated proteins 11Glu Phe His His His His His His Thr Leu Ser Leu Pro Glu Ser Gly 1 5 1015 Pro Val Thr Ile Ile Pro Pro Thr Val Lys Leu Phe His Ser Ser Cys 20 2530 Asp Pro Arg Gly Asp Ala His Ser Thr Ile Gln Leu Leu Cys Leu Val 35 4045 Ser Gly Phe Ser Pro Ala Lys Val His Val Thr Trp Leu Val Asp Gly 50 5560 Gln Glu Ala Glu Asn Leu Phe Pro Tyr Thr Thr Arg Pro Lys Arg Glu 65 7075 80 Gly Gly Gln Thr Phe Ser Leu Gln Ser Glu Val Asn Ile Thr Gln Gly 8590 95 Gln Trp Met Ser Ser Asn Thr Tyr Thr Cys His Val Lys His Asn Gly100 105 110 Ser Ile Phe Glu Asp Ser Ser Arg Lys Cys Ser Glu Ser Asp ProArg 115 120 125 Gly Val Thr Ser Tyr Leu Ser Pro Pro Ser Pro Leu Asp LeuTyr Val 130 135 140 His Lys Ala Pro Lys Ile Thr Cys Leu Val Val Asp LeuAla Thr Met 145 150 155 160 Glu Gly Met Asn Leu Thr Trp Tyr Arg Glu SerLys Glu Pro Val Asn 165 170 175 Pro Gly Pro Leu Asn Lys Lys Asp His PheAsn Gly Thr Ile Thr Val 180 185 190 Thr Ser Thr Leu Pro Val Asn Thr AsnAsp Trp Ile Glu Gly Glu Thr 195 200 205 Tyr Tyr Cys Arg Val Thr His ProHis Leu Pro Lys Asp Ile Val Arg 210 215 220 Ser Ile Ala Lys Leu Pro GlyLys Arg Leu Ala Pro Glu Val Tyr Met 225 230 235 240 Leu Pro Pro Ser ProGlu Glu Thr Gly Thr Thr Arg Thr Val Thr Cys 245 250 255 Leu Ile Arg GlyPhe Tyr Pro Ser Glu Ile Ser Val Gln Trp Leu Phe 260 265 270 Asn Asn GluGlu Asp His Thr Gly His His Thr Thr Thr Arg Pro Gln 275 280 285 Lys AspHis Gly Thr Asp Pro Ser Phe Phe Leu Tyr Ser Arg Met Leu 290 295 300 ValAsn Lys Ser Ile Trp Glu Lys Gly Asn Leu Val Thr Cys Arg Val 305 310 315320 Val His Glu Ala Leu Pro Gly Ser Arg Thr Leu Glu Lys Ser Leu His 325330 335 Tyr Ser Ala Gly Asn 340

What is claimed is:
 1. An immunogenic polypeptide, comprising a self IgEportion and a non-self IgE portion, wherein said immunogenic polypeptideis effective to induce an anti-self IgE response in a mammal.
 2. Theimmunogenic polypeptide of claim 1, wherein said mammal is a human. 3.The immunogenic polypeptide of claim 1, wherein said self portioncomprises at least a portion of a CH3 domain of IgE.
 4. The immunogenicpolypeptide of claim 1, wherein said polypeptide is capable ofdimerizing to form a soluble immunogenic dimer effective to induce saidanti-self IgE response in said mammal.
 5. The immunogenic polypeptide ofclaim 1, wherein said non-self IgE portion comprises a first region anda second region, said self IgE portion being located between said firstand second regions of said non-self IgE portion.
 6. The immunogenicpolypeptide of claim 5, wherein said first region comprises at least aportion of an IgE CH2 domain.
 7. The immunogenic polypeptide of claim 5,wherein said second region comprises at least a portion of an IgE CH4domain.
 8. The immunogenic polypeptide of claim 1, wherein said non-selfIgE portion comprises an IgE sequence present in a non-placental mammal.9. The immunogenic polypeptide of claim 8, wherein said non-placentalmammal is selected from the group consisting of opossum, platypus,koala, kangaroo, wallaby, and wombat.
 10. The immunogenic polypeptide ofclaim 1, wherein said self IgE portion lacks the CH2 domain of an IgEantibody.
 11. The immunogenic polypeptide of claim 1, wherein saidanti-self IgE response is a polyclonal response.
 12. A vaccine complexfor vaccinating a mammal, said complex comprising a first and secondpolypeptide, wherein each of said first and second polypeptides containsat least two similar amino acid sequences at least five amino acidresidues in length, wherein said first and second polypeptides areconnected to form said complex, and wherein administration of saidcomplex to said mammal induces an immune response against at least aportion of said first or second polypeptide.
 13. The complex of claim12, wherein said first or second polypeptide comprises an amino acidsequence expressed by said mammal.
 14. The complex of claim 12, whereinsaid first and second polypeptides are identical.
 15. The complex ofclaim 12, wherein the connection of said first and second polypeptidescomprises a disulfide bond.
 16. The complex of claim 12, wherein theconnection of said first and second polypeptides comprises anon-covalent interaction.
 17. The complex of claim 12, wherein saidfirst or second polypeptide comprises a linker site.
 18. The complex ofclaim 12, wherein said complex comprises a linking molecule.
 19. Thecomplex of claim 18, wherein said linking molecule connects said firstand second polypeptide.
 20. The complex of claim 12, wherein saidcomplex comprises a third polypeptide, said third polypeptide having acytokine activity.
 21. The complex of claim 20, wherein said cytokineactivity is an activity of a cytokine selected from the group consistingof interferon-α, interferon-β, interferon-γ, TNF-α, IL-1, IL-2, IL-4,IL-6, IL-12, IL-15, IL-18, and granulocyte-macrophage colony stimulatingfactor.
 22. A vaccine complex for vaccinating a mammal, said complexcomprising a first polypeptide connected to a second polypeptide,wherein said first polypeptide contains at least two similar amino acidsequences at least five amino acids in length, wherein said secondpolypeptide has a cytokine activity, and wherein administration of saidcomplex to said mammal induces an immune response against at least aportion of said first polypeptide.
 23. A vaccine complex for vaccinatinga mammal, said complex comprising a first, second, and thirdpolypeptide, wherein said first, second, and third polypeptides areconnected to form said complex, wherein said first polypeptide has afirst cytokine activity, wherein said second polypeptide has a secondcytokine activity, and wherein administration of said complex to saidmammal induces an immune response against at least a portion of saidthird polypeptide.
 24. A vaccine complex for vaccinating a mammal, saidcomplex comprising a first polypeptide connected to a secondpolypeptide, wherein said first polypeptide is a polypeptide havinginterferon-α or interferon-β activity, and wherein administration ofsaid complex to said mammal induces an immune response against at leasta portion of said second polypeptide.